Fcepsilon-pe chimeric protein for targeted treatment of allergy responses a method for its production and pharmaceutical compositions containing the same

ABSTRACT

The present invention generally relates to a new approach for the therapy of allergic responses, based on targeted elimination of cells expressing the FcεRI receptor by a chimeric cytotoxin Fc 2′-3 -PE 40 . A sequence encoding amino acids 301-437 of the Fc region of the mouse IgE molecule was genetically fused to PE 40 ′—a truncated form of PE lacking the cell binding domain. The chimeric protein, produced in  E. coli , specifically and efficiently kills mouse mast cell lines expressing the FcεRI receptor, as well as primary mast cells derived from bone marrow. The present invention provides a chimeric protein for targeted elimination of FcεRI expressing cells especially useful for the therapy of allergic responses. The said chimeric protein is comprised of a cell targeting moiety for FcεRI expressing cells and a cell killing moiety. The preferred killing moiety is the bacterial toxin Pseudomonas exotoxin (PE). This Pseudomonas exotoxin is a product of  Pseudomonas aeruginosa . The present invention also relates to a method for the preparation of said protein. This chimeric protein is prepared by genetically fusing the Fc region of the mouse IgE molecule to PE 40 , a truncated form of PE lacking the cell binding domain. The present invention also provides pharmaceutical compositions, for the treatment of allergic diseases and for the treatment of hyperplasias and malignancies, comprising as an active ingredient the above mentioned chimeric protein and a conventional adjuvant product.

This application is a divisional application of U.S. application Ser.No. 09/091,645, filed on Jun. 18, 1998, which issued as U.S. Pat. No.6,919,079 on Jul. 19, 2005 and was a national stage entry ofPCT/IL96/00181, which was filed Dec. 18, 1996, and claimed priority toIsraeli Application No. 116436, which was filed Dec. 18, 1995.

FIELD OF THE INVENTION

The present invention generally relates to a novel approach for thetherapy of allergic responses. More specifically the present inventionrelates to Fcε-PE chimeric protein for targeted elimination of FcεRIexpressing cells, a method for its production, and pharmaceuticalcompositions containing the same. This chimeric protein is composed ofcell targeting which is a part of IgE molecule linked to cell killingmoieties for recognizing and destroying cells overexpressing thespecific receptor. The killing moiety used in the chimeric protein ofthe present invention is the bacterial toxin Pseudomonas exotoxin (PE)(a product of Pseudomonas aeruginosa).

BACKGROUND OF THE INVENTION

About twenty percent of the world population suffers from variousallergic diseases such as asthma, allergic rhinitis, food allergies,atopic dermatitis and anaphylaxis. The alarming increase in theprevalence of allergic diseases over the past decade has led to a clearneed for more effective treatment.

The interaction between IgE and mast cells or basophils is the primaryeffector pathway in allergic responses. IgE binds to high-affinityreceptor (FcεRI) for its constant region, found almost exclusively onthe surface of these cells. The binding itself, in spite of the lowdissociation rate, does not result in stimulation of the cell. However,cross-linkage of cell surface-bound IgE by multivalent antigen causesreceptor aggregation, triggering explosive cellular degranulationwhereby mediators of allergy such as cellular degranulation wherebymediators of allergy such as histamine and seretonin are released.

The fact that distribution of the FcεRI receptor is restricted to cellsparticipating in an allergic response makes it an attractive candidatefor targeted immunotherapy by chimeric cytotoxins. Chimeric cytotoxinsare a novel class of targeted molecules constructed by gene fusiontechniques. These molecules are composed of cell targeting and cellkilling moieties, enabling them to recognize and destroy cellsoverexpressing specific receptors.

The bacterial toxin Pseudomonas exotoxin (PE) used in chimeric proteinconstructs, is a product of Pseudomonas aeruginosa. Having accessed thecytoplasm, PE inhibits protein synthesis by its ADP-ribosylationactivity, thus causing cell death (Middlebrook, J. I., and Dorland, R.B. 1984. Bacterial toxins: cellular mechanisms of action. Microbiol.Rev. 48, 199). Effective chimeric cytotoxins have been constructed byfusion of cDNAs encoding various growth factors or single chainantibodies with PE derivatives lacking intrinsic cell binding capacity.One of these chimeric proteins designated IL₂-PE₄₀, constructed totarget and selectively eliminate activated T cells overexpressing IL₂receptors, was shown to provide effective and selectiveimmunosuppression in various models of autoimmune disorders, graftrejection and cancer (Lorberboum-Galski, H. 1994. Interleukin2-Pseudomonas exotoxin A (IL2-PE40) chimeric protein for targetedimmunotherapy and the study of immune responses. J. Toxicol.-ToxinRewiewes, 13 (1), 105).

The entire recombinant constant region of IgE (Fcε) expressed inbacteria, have an affinity for FcεRI receptor comparable to that of thenative IgE, as well as the capacity to sensitize basophils for anti-IgEindused histamine release. When recombinant fragments of human Fcεexpressed in bacteria, were tested for receptor binding, a peptidecorresponding to residues 301-376 at the junctions of domains 2 and 3 ofthe constant region was found to be sufficient for high-affinity bindingto the receptor. It was also reported that ε-chain dimerization was notrequired for receptor binding (Helm, B., Marsc, P., Vercelli, D.,Padlan, E., Gould, H., and Geha, R. 1988. The mast cell binding site onhuman immunoglobulin E. Nature 331, 180).

The present invention generally relates to a novel approach for thetherapy of allergic responses. At present the major known groups ofdrugs used in the treatment of asthma and allergic disorders are:

-   -   1. β2 agonists—produce airway dilatation through simulation of        β2 adrenergic receptors.    -   2. Methylxantines—smooth muscle relaxants, produce        bronchodilatation.    -   3. Glucocorticoids—reduce inflammation.    -   4. Cromolyn sodium—prevents mast cell degranulation.    -   5. Antihistamines—prevents histamine action on it's target        cells.

Although widely used, all of these drugs have notable disadvantages inregard to:

-   -   1. Specificity: The action of all of these drugs (except        cromolyn sodium) is not mast cell specific. Therefore, they can        not prevent the release of allergy mediators but rather reverse        or block the effects caused by their action. The treatment by        these drugs is symptomatic, it can be started only after the        onset of the allergic reaction and thus can't be used in a        prophylactic manner.    -   2. Toxicity: Being non-specific, these drugs exert their action        on various tissues and organs causing serious side effects. The        major side effect of β2 agonists is tremor, but they also cause        cardiac arrhythmias; Methylxantines stimulate the central        nervous system, causing nervousness, nausea, vomiting, anorexia,        headache and cardiac muscle-causing tachycardia. At high plasma        levels there is a danger of seizures and arrhythmias.        Antihistamines affect the central nervous system, causing        sedation. Steroids are most harmful, causing suppression of the        pituitary-adrenal function, fluid and electrolyte disturbances,        hypertension, hyperglycemia, increased susceptibility to        infections, osteoporosis and arrest of growth in children.    -   3. Duration of the effect: β-adrenergic agonists, aminoxantines        and antihistamines are mostly short-acting drugs, and as such        must be administered frequently. Steriods which are long-acting        drugs, have also long induction time and are of little value in        emergencies.

The only existing mast cell specific drug is Cromolyn sodium. This drugcan be used prophilactically, essentially without side effects. However,it has a very short half life, very long induction time, it can beapplied only locally and only part of the patients respond to it. Allthese make use of Cromolyn sodium very limited.

A number of attempts to interfere with interaction between IgE and it'shigh-affinity receptor, as a basis for anti-allergy therapy, have beenreported in recent years. Recombinant peptides comprising structuralelements from IgE (Helm, B., Kebo, D., Vercelli, D., Glovsky, M. M.,Gould, H., Ishizaka, K., Geha, R., and Ishizaka, T. 1989. Blocking thepassive sensatization of human mast cells and basophil granolocytes withIgE antibodies by a recombinant human ε-chain fragment of 76 aminoacids. Proc. Natl. Acad. Sci. USA 86, 9465.) or FCεRI (Ra, C.,Kuromitsu, S., Hirose, T., Yasuda, S., Furuichi, K., and Okumura, K.1993. Soluble human high affinity receptor for IgE abrogates theIgE-mediated allergic reaction. Int. Immunol. 5, 47.; Haak-Frendscho,M., Ridgway, J., Shields, R., Robbins, K., Gorman, C., and Jardieu, P.1993. Human IgE receptor a-chain IgG chimera blocks passive cutaneousanaphylaxis reaction in vivo. J. Immunol. 151, 351.) have beeninvestigated as competitive inhibitors of the IgE-FcεRI interaction.Monoclonal antibodies generated against IgE (Baniyash, M., and Eshhar,Z. 1984. Inhibition of IgE binding to mast cells and basophils bymonoclonal antibodies to murine IgE. Eur. J. Immunol. 14, 799) or FcεRI(Kitani, S., Kraft, D., Fischler, C., Mergenhagen, S. E., andSiraganian, R. P. 1988. Inhibition of allergic reactions with monoclonalantibody to the high affinity IgE receptor. J. Immunol. 140, 2585.),capable of blocking IgE binding to the receptor, without causing mastcell degranulation have also been tested. However, the affinity of IgEfor FcεRI is very high (K_(M)=10⁻¹⁰M), so that once it is bound to it'sreceptor, the IgE molecule remains attached to the cell membrane forseveral weeks. Moreover, mast cell can be activated at low receptoroccupancy: the cross-linkage of as few as 5% of receptors is sufficientto cause mast cell degranulation. These two properties of the systemimpede inhibition by competitive agents, thus limiting their clinicalvalue. Our anti-allergy molecule depends to a much lesser extent on theability to compete with IgE. Once having entered the target cell througha non-occupied IgE receptor, the chimeric protein affects the targetcell. Moreover, early expression of the receptor in the maturationcourse of mast calls should allow the elimination of immature targetcells before they are capable of mediator release. As the receptor isnot expressed on stem cells, no damage to bone marrow is expected on thewhole.

The IgE system is quite complex and diverse. Interactions between IgEand its binding structures have many functions apart from the allergicresponse, some of which are only beginning to emerge. Monoclonalantibodies against IL-4, the IL-4 receptor or the low-affinity IgEreceptor eliminate expression of IgE in mice but have more generalimmunosupressive effects. The advantage of the present invention inwhich the high-affinity IgE receptor is targeted and not the overall IgEsystem, is therefore evident.

SUMMARY OF THE INVENTION

The present invention generally relates to a new approach for therapy ofallergic responses, based on targeted elimination of cells expressingthe FcεRI receptor by a chimeric cytotoxin Fc_(2′-3)-PE₄₀. A sequenceencoding amino acids 301-437 of the Fc region of the mouse IgE moleculewas genetically fused to PE₄₀—a truncated form of PE lacking the cellbinding domain. The chimeric protein, produced in E. coli, specificallyand efficiently kills mouse mast cell lines expressing the FcεRIreceptor, as well as primary mast cells derived from bone marrow.

The present invention provides a chimeric protein for targetedelimination of FcεRI expressing cells especially useful for the therapyof allergic responses. The said chimeric protein is comprised of a celltargeting moiety for the FcεRI expressing cells and a cell killingmoiety. The preferred killing moiety is the bacterial toxin Pseudomonasexotoxin (PE). This Pseudomonas exotoxin is a product of Pseudomonasaeruginosa.

The present invention also relates to a method for the preparation ofsaid protein. This chimeric protein is prepared by genetically fusingthe Fc region of the mouse IgE molecule to PE₄₀, a truncated form of PElacking the cell binding domain.

The present invention also provides a pharmaceutical compositions, forthe treatment of allergic diseases and for the treatment of hyperplasiasand malignancies, comprising as an active ingredient the above mentionedchimeric protein and a conventional adjuvant product.

The present invention further relates to the method for the preparationof these pharmaceutical compositions comprising genetically fused Fcregion of the mouse IgE molecule to PE₄₀ and adding, if needed, aconventional adjuvant product. The pharmaceutical compositions accordingto the present invention may be in any suitable form for injection, fortopical application, or for oral administration.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: Schematic representation of plasmids coding for theFc_(2′-3)-PE₄₀ and Fc₂₋₄-PE₄₀ chimeric proteins.

FIG. 2: SDS-polyacryamide gel electrophoresis analysis of cell fractionscontaining Fcε-PB₄₀ chimeric proteins. Samples containing 5 μg. totalprotein were loaded onto 10% gels. (A) Cells expressing Fc_(2′-3)-Pe₄₀Lane 1, markers; Lane 2 whole cell extract; Lane 3, soluble fraction;Lane 4, insoluble fraction. (B) Cells expressing Fc₂₋₄-PE₄₀. Fractionsare as described in A.

FIG. 3: Immunobloting of insoluble fractions containing Fcε-PE₄₀chimeric proteins with antibodies against PB (A) and IgB (B). Samplescontaining 1 μg of total protein were loaded onto 10% SDS-polyacrylamidegels. Electrophoressed samples were transferred onto nitrocellulose andprocessed as described in Materials and Methods. (A) αPE: Lane 1,Fc_(2′-3)-PE₄₀; Lane 2: Fc₂₋₄-PE₄₀ (B) αIgE: Lanes are as described inA.

FIG. 4: Cytotoxic activity of various chimeric proteins against MC-9cells (A), C57 cells (B), and Abelson cells (C). Cells were incubatedfor 20 h with insoluble fractions containing the chimeric proteins-▪-Fc_(2′-3)-PE₄₀; -●- Fc₂₋₄-PB₄₀; -▴- Fc_(2′-3)-PE_(40M); or -□- PE₄₀at various concentrations (according to total protein concentration).Experiments with MC-9 cells were performed in the presence of IL₃ (20u/ml) and IL₄ (10 u/ml). [³H] Leucine incorporation into cell proteinswas measured as described in Materials and Methods. The results areexpressed as the percentage of protein synthesis of control cells notexposed to chimeric proteins.

FIG. 5: Inhibition of Fc_(2′-3)-PE₄₀ cytotoxicity by (A) IgE and (B)αPE. Cells were incubated with whole IgE (40 mg/ml) for 1 h at 4° C.before the addition of Fc_(2′-3)-PE₄₀ αPE (10 mg/ml) was added a fewminutes prior to the addition of Fc_(2′-3)-PE₄₀. All other experimentalconditions were as described in FIG. 4. ▪

FIG. 6: Cytotoxic activity of various chimeric proteins against bonemarrow derived primary mast cells (BMMC). Bone marrow was cultured asdescribed in Materials and Methods. Experiments were performed on the16^(th) day of culture, as described in FIG. 4, in the presence of IL₃(20 u/ml) and IL4 (10 u/ml).

FIG. 7: Cytotoxic activity of various chimeric proteins against the C57cells in the presence of: αFcγRII/III (2.4G2). Cells were incubated with2.4G2 (50 μg/ml) or galactose (25 mm) for 30 mm. at 37° C. prior to theaddition of Fc_(2′-3)-PE₄₀. All other experimental conditions were asdescribed in FIG. 4. (A) Fc_(2′-3)-PE₄₀ in the absence (-●-) or presence(-◯-) of 2.4G2. (B): Fc_(2′-3)-PE₄₀ in the absence (-Δ-) or presence(-▴-) of galactose.

FIG. 8: Cytotoxic activity of various chimeric proteins against FcεRIIbearing cells. (-◯-) B splenocytes.-▪-0.12A3 B cell hybridoma. Bsplenocytes were preincubated for 16 h. with LPS (50 μg/ml) and IL₄ (50u/ml). All other experimental conditions were as described in FIG. 4.

FIG. 9(A): The effect of Fc_(2′-3)-PE₄₀ on seretonin release from C57cells. Cells were labeled overnight with [³H] Hydroxytryptaminecreatinine sulfate. The cells were then washed and incubated withFc_(2′-3)-PE₄₀ (10 μg/ml). Control cells were not exposed to anyprotein. At different time points [³H] Hydroxytryptamine creatininesulfate release into the medium was measured.-▪-control, -

- Fc_(2′-3)-PE₄₀

FIG. 9(B): Time-dependant cytotoxycity of Fc_(2′-3)-PE₄₀ against C57cells. Unlabeled cells were incubated as in (A). At the same timepoints, cells were pulsed for 1 h with [³H] Leucine and itsincorporation into cellular proteins was measured. The results areexpressed as the percentage of protein synthesis of control cells notexposed to chemeric proteins.

FIG. 10: Immunoblotting of Fc_(2′-3)-PE₄₀ chimeric proteinelectrophoresed under the following conditions with anti-PE: A) in SDSunder reducing conditions, B) in SDS under nonreducing conditions and C)a nondenaturing gel (i.e. no reduction, no SDS).

FIG. 11: Internalization of Fc_(2′-3)-PE₄₀ chimeric protein by MC-9cells. Samples containing 20 μl of each of the following fractions wereloaded onto SDS-10% polyacrylamide gels: lane 1, 40 ng Fc_(2′-3)-PE₄₀;lane 2, supernatant of the cells; lane 3, last wash before the acidtreatment; lane 4, acid wash supernatant; lane 5, last wash after acidtreatment; and lane 6, lysed cells.

FIG. 12(A): The effect of Fc_(2′-3)-PE₄₀ on serotonin release from C57cells. A) Cells were labeled overnight with [³H] hydroxytryptaminecreatinine sulfate. The cells were then washed and exposed to variousconcentrations of Fc_(2′-3)-PE₄₀ for 30 minutes. Control cells werepre-incubated with IgE and exposed to DNP and [³H] hydroxytryptaminecreatinine sulfate released into the medium was measured:

▪ Control,

IgE-DNP, ▪ 100 ng,

250,

1000 ng, or □5000 ng Fc_(2′-3)-PE₄₀

FIG. 12(B): Cells were incubated with Fc_(2′-3)-PE₄₀ at different timepoints [³H] hydroxytryptamine sulfate release into the medium wasmeasured; legends as in FIG. 12(A).

FIG. 12(C): Time dependent cytotoxicity of Fc_(2′-3)-PE₄₀ against C57cells. Unlabeled cells were incubated as in FIG. 12(B). At the same timepoints cells were pulsed for 1 h with [³H] leucine and its incorporationinto cellular proteins was measured. The results are expressed as thepercentage of protein synthesis of control cells not exposed to chimericproteins.

DETAILED DESCRIPTION OF THE INVENTION

The Fc-PE chimeric protein according to the present invention has anumber of advantages over the existing known drugs:

-   -   1. Specificity: Fc-PE is highly specific, affecting the cells        (mast cells and basophils) responsible for the release of        allergic mediators. As it prevents the allergic attack, it can        be of great value as a prophylactic treatment.    -   2. Toxicity: As it acts on affector cells and not on it's target        organs, Fc-PE is expected to have little, if any, side effects.        Moreover, as the receptor is not expressed on stem cells, no        damage to bone marrow and immunosuppression are anticipated.        Re-institution of a normal physiological state is expected to        occur within several weeks after the end of the treatment.

3. Duration of the effect: Because maturation of mast cells takesseveral weeks, the effect of Fc-PE is predicated to be long-standing,eliminating the need for frequent administration. Moreover, as in vitrostudies indicate that reduction of 80% in cellular protein synthesis isobserved in less than 4 hours, induction time of Fc-PE is expected to berelatively short, enabling it's usage in acute phase allergic reactions.

Fcε-PE can also be valuable in the treatment of hyperplasias andmalignancies of mast cells and basophils, like systemic mastocytosis (inboth benign and malignant forms) and basophilic leukemia. Chemotherapyis not appropriate for patients with benign mastocytosis due to severeside effects. On the other hand, there is no good clinical protocol forthe treatment of the malignant diseases. Fcε-PE chimeric protein, beinghighly potent and selective can be used for both benign and malignantconditions involving cells expressing the FcεRI receptors.

The following experimental results indicate that the Fc_(2′-3-PE)40chimeric protein according to the present invention is a promisingcandidate for effective and selective allergy therapy.

The present invention provides a FCε-PE chimeric cytotoxin protein forthe targeted elimination of FcεRI expressing cells, useful especiallyfor the therapy of allergic responses such as asthma, allergic rhinitis,food allergies, atopic dermatitis, and anaphylaxis.

The said invention will be further described in detail by the followingexperiments. These experiments do not intend to limit the scope of theinvention but to demonstrate and clarify it only.

1. Construction of Fcε-PE₄₀ Chimeric Proteins.

For the targeting moiety of the chimeric proteins fragments of the mouseIgE constant region (Fcε) are used as it binds both to human and tomouse high affinity IgE receptors (Conrad, D. H., Wingard, J. R., andIshizaka, T. 1983 The interaction of human and rodent IgE with the humanbasophil IgE receptor. J. Immunol. 130, 327).

We used a sequence corresponding to a.a. 301-437, containing the COOHterminus of domain 2 and the entire domain 3(C₂′-C₃). We used also asequence corresponding to a.a. 225-552, containing the wholeC₂-C₄domains. The cDNA for these fragments was obtained by RT-PCR, usingRNA isolated from mouse B cells which were isotopically switched tosecrete IgE and a specific set of primers. B cells obtained from thespleen of a 6-week-old BALB/C mouse were separated by negative selectionusing anti-Thy1.2 and rabbit complement. Cells were incubated at 2×10⁶cells/ml in the presence of Lipopolysaccharide (LPS, 10 μg/ml) and IL₄(500 u/ml) for 5 days to induce isotypic switching for IgE production.After 5 days, total cellular RNA was isolated (RNAzol TM B isolation kitproduced by BIOTECK Laboratories, Houston, USA.). Total RNA (2.5 μg) wasthen reverse transcribed into first strand cDNA, using the reversetranscription System (Promega, USA) under conditions, recommended by themanufacturer. The cDNA was diluted to a total volume of 1 ml with TEbuffer (10 mM Tris-HCL, pH 7.6, 1 mM EDTA) and stored at 4° C. untilused.

FCε fragments were generated by PCR, using cDNA and a pair of syntheticoligonucleotide primers 5′-GCG GAT CCC ATA TGG AGC AAT GGA TGT CGT-3′(sense, starting from nucleotide 406, according to gene bank sequenceJ00476), SEQ ID NO: 5, and 5′-GCG GAT CCC ATA TGT GGG GTC TTG GTG ATGGAA C-3′ (antisense, starting from nucleotide 813) for the FCε_(2′-3)sequence, SEQ ID NO: 6, and 5′-GCG GAT CCC ATA TGC GAC CTG TCA ACA TCACTG-3′ (sense, starting from nucleotide 175), SEQ ID NO: 7, and 5′-GCGGAT CCC ATA TGG GAG GGA CGG AGG GAG G-3′ (antisense, starting fromnucleotide 1167) for the FCs24 sequence, SEQ ID. NO: 8.

Synthetic oligonucleotides were synthesized on an Applied Biosystems DNAsynthesizer and purified on oligonucleotide purification cartridges. Thevent polymerase enzyme (Biolabs) was used for amplification. Thereaction mixture was incubated in a DNA thermal cycler (MJ Research,Inc., USA.) for 33 cycles. Each cycle consisted of 1 mm. at 95° C., 1mm. at the annealing temperature and 2 mm. at 72° C. The MgSO₄concentration and the annealing temperature used for each primer pairwere: 2.5 mM and 61° C. for FC_(2′-3′), 2 mM and 57° C. for FC₂₋₄.

The pHL 906 plasmid, which encodes IL₂-PE₄₀, was described previously(Fishman, A., Bar-Kana, Y., Steinberger, I., and Lorberboum-Galski, H.1994. Increased cytotoxicity of IL2-PE chimeric proteins containingtargeting signal for lysosomal membranes. Biochem. 33, 6235). The pHL906plasmid was cut with Ndel, obtaining the larger fragment of 3596 bp. Theabove Fcε fragment was inserted into the Ndel site of pHL906. Theresulting plasmids, pAF2302 and pAF2415, coding for the C₂′-C₃ and C₂-C₄fragments respectively, each fused 5′ to PE₄₀, were characterized byrestriction and sequence analysis (results not shown). Escherichia colistrain HB101 was used for transformation and preparation of theplasmids.

2. Expression and Partial Purification of the Chimeric Proteins.

The newly designed chimeric protein, Fcε-PE₄₀ encoded by plasmid pAF2302was expressed in E. coli strain BL21(lambda-DE3) which carries a T7 RNApolymerase gene in a lysogenic and inducible form. Induction wasperformed at O.D.₆₀₀0.5 or 180 min. in the presence of isopropylβ-D-thiogalactoside (IPTG, 1 mM final concentration). A pelletexpressing cells was suspended in TE buffer (50 mM Tris pH 8.0, 1 mMEDTA) containing 0.2 mg/ml lysosyme, sonicated (three 30 s bursts) andcentrifuged at 30,000×g for 30 min. The supernatant (soluble fraction)was removed and kept for analysis. The pellet was denatured inextraction buffer (6 M guanidine-hydrochloride, 0.1 M Tris pH 8.6, 1 mMEDTA, 0.05 M NaCl and 10 mM DTT) and stirred for 30 min. at 4° C. Thesuspension was cleared by centrifugation at 30,000×g for 15 min. and thepellet discarded. The supernatant was; then dialysed against 0.1 M Tris(pH 8.0), 1 mM EDTA, 0.25 mM NaCl and 0.25 mM L-Arginine for 16 h. Thedialysate was centrifuged at 15,0000×g for 15 min. and the resultantsupernatant (insoluble fraction, guanidine-hydrochloride treated) wasused as a source of the chimeric proteins. Proteins were characterizedby gel electrophoresis (FIG. 2). The protein profile of whole cellextracts revealed the high expression level of the chimeric protein.

The protein was further characterized by Western blot analysis usingantibodies against PE (FIG. 3A) and against IgE (Serotec, England) (FIG.3B). The electrophoresed samples were transferred onto nitrocelluloseand immunoblotted as described (Lorberboum-Galski, H., Fitzgerald, D.J., Chaudhary, V., Ashya, S., and Pastan, I. 1988. Cytotoxic activity ofan interleukin 2 —Pseudomonas exotoxin chimeric protein produced inEscherichia coli. Proc. Natl. Acad. Sci. USA 85, 1992). A Vectastain ABCKit (Vector Laboratories, USA) was used according to the manufacturer'sinstructions. The chimera reacted with both antibodies, thus confirmingthe cloning and production of in-frame full-length chimeric protein.

Subcellular fractionation of expressing cells revealed that theinsoluble fraction (inclusion bodies) was particularly rich withchimeric protein (FIG. 2). This fraction was therefore used as thesource of the chimeric protein.

The ADP-ribosylation activity of tested samples was measured using wheatgerm extracts enriched in elongation factor 2 as substrate, as describedpreviously, and revealed that the novel chimeric protein wasenzymatically active (results not shown).

3. Effect of Fc_(2′-3)-PE₄₀ Chimeric Protein on Mouse Mast Cell Lines.

The cytotoxic effect of the chimeric protein was tested on various mousemast cell lines known to express the FcεRI receptor. The cytotoxicactivity of the chimeric protein was evaluated by inhibition of proteinsynthesis, as measured by [³H] Leucine incorporation. Variousconcentrations of the chimeric protein, diluted with 0.25% bovine serumalbumin in phosphate-buffered saline, were added to 2×10⁴ cells/0.2 mlseeded in 96-well plates for 20 h., followed by an 8 h pulse with 2 μCiof [³H]-Leucine. The results are expressed as a percentage of thecontrol experiments in which the cells were not exposed to the chimericprotein. All assays were carried out in triplicate in three separateexperiments.

Three target cell lines expressing the FcεRI receptor were used: MC-9, amast cell line originating in mouse fetal liver and dependent on IL₃ forgrowth, C57, an IL₃ independent mast cell line originating in mouse bonemarrow; and the Abelson-virus transformed mast cell line originating inmouse midgestation embryonic placenta.

Fcε-PE₄₀ was found to be cytotoxic in a dose-dependent manner to all thecell lines tested (FIG. 4). The MC-9 and C57 lines were extremelysensitive to the chimeric toxin, with an ID₅₀ of 50-75 ng/ml and 100-125ng/ml, respectively. The Alelson cell line was much less sensitive (ID₅₀of 1200-1500 ng/ml).

4. Specificity of FCε-PE₄₀ Response.

To verify the specificity of Fc_(2′-3)PE₄₀ activity, two controlproteins, PE₄₀ and Fc_(2′-3)-PE_(40M), were generated and evaluated fortheir effect on target and non target cells. To constructFc_(2′-3)-PE_(40M), the region coding for the 122 amino acids at theC-terminal of PE was exised with EcoRI and BamHI and replaced by acorresponding fragment carrying a deletion at amino acid 553.

PE₄₀, which has no intrinsic targeting capacity had, as expected, noeffect on the target cell lines (FIG. 4). Fc_(2′-3)-PE_(40M) whichpossesses a Fc_(2′-3) moiety linked to a mutated, enzymatically inactiveform PE₄₀, was also not cytotoxic to the target cells (FIG. 4).

In addition, it was possible to block the cytotoxic effect ofFc_(2′-3)-PE₄₀ against target cells by whole mouse IgE (40 μg/ml, FIG.5A) or by a αPE polyclonal antibody (10 μg/ml, FIG. 5B).

The effect of Fc_(2′-3)-PE₄₀ was also tested on various mouse non-targetcell lines (Table 1). All cell lines of hemopoetic origin wereunaffected by the chimeric protein. Suprisingly, fibroblast and hematomacell lines exhibited some sensitivity to chimeric toxin, although theID₅₀ values were twenty-fold higher than those of the MC-9 cells (Table1).

The above data demonstrates that the toxic effect of Fc_(2′-3-PE)40 onmast cell lines is due to a specific response mediated by the Fc_(2′-3)moiety which targets the cytotoxic part of the chimera (PE₄₀) into thecell.

5. Effect of Chimeric Proteins on Primary Mast Cells.

As it is likely that fresh murine mast cells react differently fromestablished cell lines, we also tested primary mast cells obtained fromnormal mice for their sensitivity to Fc_(2′-3)-PE₄₀. When cultured inthe presence of IL₃ for two weeks, mouse bone marrow differentiates intoan almost pure population of cells with the morphology of immature mastcells, containing granules and expressing the FcεRI receptor.

BALB/C mice aged 4-6 weeks were sacrificed and their bone marrow wasaseptically flushed from femurs into 0.9% cold NaCl. The cell suspensionwas washed twice with 0.9% NaCl, centrifuged for 10 min. at 300×g andfinally resuspended in RPMI 1640 medium containing 10% heat inactivatedfetal calf serum, 4 mM L-glutamine, 1 mM sodium piruvate, 0.1 mMnonessential amino acids, 5×10⁻⁵ M β-mercaptoethanol, 100 u/mlpenicillin, 100 μg/ml streptomycin and 20 u/ml recombinant mouse IL₃.Cells were grown in tissue culture flasks at a density of 10⁶ cells/ml,at 37° C. in a 5% CO₂ humidified atmosphere for 2-3 weeks. The mediawere changed every 7 days. Recombinant IL₄ (10 u/ml) was added startingfrom day 7 in culture.

To follow the degree of maturation, cells were mounted on slides,stained with acidic Toluidine Blue (pH 1.0) and examined microscopicallyunder oil.

The effect of chimeric proteins was tested on bone marrow derived mastcells (BMMC) on the 16th day of cultures. As shown in FIG. 6,Fc_(2′-3)-PE₄₀ was cytotoxic to BMMC in a dose dependent manner, with anID₅₀ of 125 ng/ml. At a high chimeric protein dose, there was nearly100% inhibition of protein synthesis. None of the control proteinsFc_(2′-3)-PE_(40M) or PE₄₀ displayed cytotoxicity against BMMC (FIG. 6).Thus, primary mast cells respond towards the chimeric protein similarlyto the established mast cell lines (FIGS. 4 and 6).

6. Receptor Specificity of Fc_(2′-3)-PE₄₀.

Aside from the high affinity FcεRI receptor, three other membranesurface structures were reported to bind IgE with low affinity—the lowaffinity FcεRII receptor, the εBP galactoside-binding protein (alsotermed MAC-2 or CBP35) and the FcγRII/III receptor. These structuresappear on various cell types, mainly of hemopoethic origin, but also onfibroblasts (εBP). FcγRII/III and εBP appear on mast cell membranes inaddition to FcεRI. As our aim was to target only mast cells, it wasessential to prove that the chimeric protein does not recognize thesestructures and thus can not be internalized through them. Theoreticallyour chimeric protein does not fulfill the binding requirements of thelow-affinity IgE binding structure FcεRII, εBP and FcγRII/III. FcεRIIbinds only disulfide linked ε-chain dimmers, while our protein lacksdomain 4 which is essential for dimerization. εBP binds onlyglycosylated IgE; Fc_(2′-3)-PE₄₀ being produced in bacteria, is notglycosylated. FcγRII/III binds IgE-immunocomplexes but not free IgE.Nevertheless, the issue of receptor binding was challengedexperimentally.

Experiments involving εBP and FcγRII/III were performed on C57 mastcells, known to express these receptors in addition to FcεRI. To testwhether the chimeric protein can enter the cell via the FcγRII/IIIreceptors, cells were preincubated with the 2.4G2 antibody (Pharmigen)(50 μg/m) prior to addition of the chimeric protein. This monoclonalantibody, which binds to the extracellular domains of both FcγRII andthe FcγRIII receptors was shown to be a competitive inhibitor of IgEbinding. As can be seen in FIG. 7A, there was no difference in thecellular response to Fc_(2′-3)-PE₄₀ between control cells and cellspreincubated with the antibody.

We next examined whether εBP is involved in the cytotoxicity ofFc_(2′-3)-PE₄₀. As εBP is attached to membrane carbohydratedeterminants, addition of lactose to the culture medium causes itsdissociation from the cell surface. We found no difference in thecellular response to Fc_(2′-3)-PE₄₀ in the presence or absence oflactose (25 mM, FIG. 7B).

Additional experiments in the presence of 2.4G2 antibody and lactosewere performed on fibroblast cell lines that were found partiallyresponsive to the chimeric protein (Table 1). Again, there was nodifference in FC_(2′-3)-PE₄₀ cytotoxicity against treated and controlcells (results not shown).

To test whether Fc_(2′-3)-PE₄₀ affects FcεRII-bearing cells, we used the0.12A3 cell line, a mouse B cell hybridoma expressing the FcεRIIreceptor. The 0.12A3 cells were totally non responsive toFc_(2′-3)-PE₄₀, even at high doses (>5000 ng/ml, FIG. 8A). As this lineloses the receptor upon long term culture, the assay was followed byFACS analysis with the B3B4 antibody against the receptor (Pharmigin).The results showed that the receptor was expressed on 54% of the cells(results not shown).

An additional experiment was performed on fresh mouse B splenocytespreincubated for 16 h. with LPS (50 μg/ml) to stimulate expression ofFcεRII. Fc_(2′-3)-PE₄₀ has no effect on these B splenocytes (FIG. 8B),although 69% of the cells expressed the receptor, as determined by FACSanalysis.

Collectively, these results suggest that Fc_(2′-3)-PE₄₀ does not bind tothe low affinity IgE-binding structures, namely FcεRII, FcγRII/III andεBP.

7. Effect of Fc_(2′-3)-PE₄₀ on Cellular Degranulation.

Because of the possible clinical applicability of Fc_(2′-3)-PE₄₀, it wasimportant to test whether treatment of mast cells with Fc_(2′-3)-PE₄₀results in the release of allergic mediators triggered upon FcεRIbinding by the chimetric protein.

C57 cells prelabelled overnight with [³H]-hydroxytryptamine (10 μci/ml)were washed, plated at 2×10⁵ cells/well in DMEM containing 10% FCS, in96-well tissue culture plates and incubated with Fc_(2′-3)-PE₄₀ (10μg/ml) at 37° C. At various time points, supernatants were separated andrelease of seretonin into the supernatant was measured. Unlabled cellswere also incubated with Fc_(2′-3)-PE₄₀ and at the same time intervalswere pulsed 1 hr with [³H] leucine to measure protein synthesisinhibition by chimeric toxin. There was no difference in supernatant[³H] seretonin content between Fc_(2′-3)-PE₄₀ treated and untreatedcells at ½, 4 or 8 hr following chimeric protein addition (FIG. 9A).Inhibition of protein synthesis reached 80% at 4 h. and a value of 90%by 8 h. (FIG. 9B). These results suggest that Fc_(2′-3)-PE₄₀ does notcause release of allergic mediators during receptor binding or uponinhibition of protein synthesis.

8. Electrophoretic Characterization of Fcε-PE40

Western blot analysis of electrophoresed samples run under non-reducingconditions (omitting 2-mercaptoethanol from the sample buffer) revealedthat the Fc2′-3-PE40 chimeric protein is predominantly present as amonomer (FIG. 10 b). For native PAGE, 2-mercaptoethanol was omitted fromthe sample buffer and the samples were not heated. In addition, SDS wasreplaced with equivalent volumes of water in the gel, sample buffer andelectrode running buffer. Under non-denaturing conditions the chimericprotein runs as a broad band (FIG. 10 c). A single native system can notdistinguish the effects of molecular weight, charge and conformation onprotein electrophoretic mobilities. However, the proximity of themolecules in the band indicates that they can not differ much in theseparameters.

9. Internalization Assay

In vitro activity of the chimeric protein is achieved only upon it'sinternalization. To rest whether the chimeric protein is internalysed,5×10⁵ cells/3 ml were incubated for 1 hour with 20 μg of the chimericprotein at 37° C. After 3 washes with cold PBS the pellet was treatedwith 0.5 ml of acid solution (0.15M NaCl, 0.15M acetic acid (pH 3)) for3 min on ice to remove membrane-bounded chimeric protein. The pH wasthen neutrilised by addition of 50% FCS following by three washed withRPMI/10% FCS. The cell pellet was lysed with 0.3 ml of RIPA lysis buffer(150 mM NaCl, 1 mM EDTA, 20 mM tris-HCl pH 7.4, 1 mMphenylmethylsulfonyl fluoride, 15% SDS, 1% deoxycholyc acid, 1% NonidetP-40). Various samples were electrophoresed and immunoblotted using α-PEand the ECL detection system (Amersham). Western blot analysis revealedundoubtfully that Fc2′-3-PE40 chimeric protein is internalized into thetarget cells (FIG. 11).

10. Effect of Fc_(2′-3)-PE₄₀ on Cellular Degranulation

C57 cells were incubated overnight with [³H]-Hydroxytryptamine (10μci/ml) at 37° C. Cells were washed 3 times to remove free[3H]-Hydroxytryptamine, plated in Tyrod's buffer (10 mM Hepes pH 7.4,130 mM NaCl, 5 mM KCl, 5.6 mM Glucose, 0.5% BSA) at 2.5×10⁵ cells/0.5 mlin 24 well tissue culture plates and incubated with IgE (10 μg/ml) for 1hour at 4° C. MgCl₂ and CaCl₂ were then added to the final concentrationof 1 mM and 1.6 mM respectively, following by incubation withDinitrophenyl-human serum albumin (DNP-HSA, 50 ng/ml) for 30 minutes orwith the different concentrations of chimeric protein for various timesat 37° C. Cell-free supernatants were collected by centrifugation andamount of [³H]-Hydroxytryptamine released was measured. No degranulationwas observed with any concentration of chimeric protein tested (FIG. 12a). As a control, cells preincubated with IgE were exposed to DNP underthe same conditions. The effect of triggering degranulation by DNP isclearly visible (FIG. 12 a). Fc_(2′-3)-PE₄₀ did not cause anydegranulation also at later stages of it's interaction with the targetcell (FIG. 12 b), while it inhibits protein synthesis by over 80% (FIG.12 c). Our results demonstrate that Fc_(2′-3)-PE₄₀ _(—) does not_triggerdegranulation at any stage during it's interaction with the cell.

TABLE 1 Cytotoxicity of Fc_(2′-3)-PE₄₀ chimeric protein against variousmouse cells Cell line Cell Origin ID₅₀ (ng/ml) TARGET MC-9 Mast cells 50-100 CELLS C57 Mast cells 100-125 BMMC Primary bone marrow-derivedmast cells Abelson Transformed mast 1,200-1,500 cells NON- HEMOPETICL₁₀A B cell, >10,000 TARGET non-secreting CELLS X₁₆B B cell, >10,000non-secreting UT B cell, >10,000 non-secreting PD1.1 T cell,immature >10,000 EL-4 T cell, mature >10,000 Erythro- >10,000 leukemiaCONNECTIVE LTK Fibroblast 1900 TISSUE Hepatoma 1500

1. A chimeric protein for therapy of allergic responses by targetedelimination of FCεRI expressing cells, the chimeric protein comprising acell targeting moiety consisting of an Fc region of an IgE moleculegenetically fused to a cell killing moiety, wherein the chimeric proteinis cytotoxic to mouse bone marrow-derived mast cells and the chimericprotein has an ID₅₀ of 125 ng/ml.
 2. A chimeric protein for therapy ofallergic responses by targeted elimination of FCεRI expressing cells,the chimeric protein comprising a cell targeting moiety consisting of anFc region of an IgE molecule genetically fused to a cell killing moiety,wherein the chimeric protein is cytotoxic to mouse bone marrow-derivedmast cells and the chimeric protein has an ID₅₀ of 100-125 ng/ml.
 3. Thechimeric protein of claim 1, wherein the chimeric protein does not bindto FCεRII, FcγRII/III or εBP.
 4. The method of claim 2, wherein thechimeric protein does not bind to FCεRII, FcγRII/III or εBP.